The genetic element flanking the Bacteroides fragilis pathogenicity island [BfPAI] in enterotoxigenic B . fragilis [ETBF] strain 86-5443-2-2 and a related genetic element in NCTC 9343 were characterized . The results suggested that these genetic elementsare members of a new family of conjugative transposons [CTns]not described previously . These putative CTns, designated CTn86and CTn9343 for ETBF 86-5443-2-2 and NCTC 9343, respectively,differ from previously described Bacteroides species CTns ina number of ways . These new transposons do not carry tetQ, andthe excision from the chromosome to form a circular intermediateis not regulated by tetracycline; they are predicted to differin their mechanism of transposition; and their sequences havevery limited similarity with CTnDOT or other described CTns.CTn9343 is 64,229 bp in length, contains 61 potential open readingframes, and both ends contain IS21 transposases . Colony blothybridization, PCR, and sequence analysis indicated that CTn86has the same structure as CTn9343 except that CTn86 lacks a 7-kb region containing truncated integrase [int2] and rteA genes and it contains the BfPAI integrated between the mob region and the bfmC gene . If these putative CTns were to be demonstrated to be transmissible, this would suggest that the bft gene can be transferred from ETBF to nontoxigenic B . fragilis strains by a mechanism similar to that for the spread of antibiotic resistance genes.

 
Enterotoxigenic Bacteroides fragilis [ETBF] has been associated with diarrheal disease in livestock, young children, and adults [22, 23, 29, 30, 33, 41, 47] . The only recognized virulencefactor of these strains is a toxin termed B . fragilis toxin,or BFT . BFT has been characterized as a 20-kDa zinc-dependentmetalloprotease [19] that mediates the cleavage of E-cadherin,resulting in an altered morphology of certain human intestinalcarcinoma cell lines [particularly HT29/C1cells], fluid accumulationin ligated lamb ileal loops, and intestinal epithelial cellproliferation [4, 14, 21, 23, 25, 36, 41, 44, 45].

It has been reported that the bft gene is contained in a 6-kb pathogenicity island termed the B . fragilis pathogenicity island, or BfPAI [9, 20] . In addition to the BfPAI, 12 kb of its flankingDNA has been sequenced [9; A . Franco and C . L . Sears, unpublisheddata] . This work revealed that the BfPAI is flanked by genesencoding putative mobilization proteins [9] . The left end ofthe BfPAI is flanked by a typical Bacteroides mobilizable region[11, 37], that is, the genes for DNA-processing enzymes [bfmA and bfmB] and the cis-acting oriT located adjacent to this genes.The region flanking the right end of the BfPAI contains a gene[bfmC] whose predicted protein shares significant identity tothe TraG family proteins for mobilization and VirD4, a proteincomponent of type IV secretion systems [9].

Colony blot analysis of a collection of ETBF and nontoxigenic B . fragilis [NTBF] strains indicated that there are three major populations of B . fragilis strains based on the presence of the BfPAI and its flanking regions: [i] pattern I strains, containing the BfPAI and its flanking region [all are ETBF strains]; [ii] pattern II strains, lacking the BfPAI and its flanking regions[all are NTBF strains]; and [iii] pattern III strains, containingthe flanking region but lacking the BfPAI [all are NTBF strains][9] . It has also been reported that the region flanking theBfPAI and a 700-bp region upstream of bft are crucial to maximalBFT production by ETBF strains [10].

The G+C contents of the BfPAI [35%] and the flanking DNA [47to 50%] differ greatly from that reported for the B . fragilis chromosome [43%] [http://www.sanger.ac.uk/Projects/B_fragilis], suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from different organisms . Basedon these results, we hypothesized that ETBF strains may haveevolved by horizontal transfer of these two genetic elementsinto a pattern II NTBF strain [9].

Many strains of Bacteroides species carry large self-transmissible elements called conjugative transposons [CTns] . CTns are genetic elements that move from the genome of a donor bacterium to the genome of a recipient bacterium by a process that requires intercellular contact . The CTn initiates conjugal transfer by excising from the chromosome to form a circular intermediate . This intermediateis nicked at the oriT, and a single-stranded copy is transferred to the recipient cell, recircularized, and integrated into the recipient [5, 7, 28, 35] . Strains of Bacteroides species carry CTns that belong to at least two distinct families; the best described is CTnDOT and its relatives [31, 37] . Many of thesetransposons confer resistance to tetracycline that is determinedby tetQ . Excision of CTnDOT and formation of the circular intermediateare stimulated 1,000- to 10,000-fold by tetracycline [31, 38].

In this study, our analyses suggest that the genetic element flanking the BfPAI in ETBF strains and a related element presentin pattern III NTBF strains are novel CTns not described previouslyin Bacteroides species.

 
Bacterial strains and growth conditions. The B . fragilis strains used in this study are described inTable 1. Bacteroides thetaiotaomicron strains BT4107 and BT400were a gift from N . B . Shoemaker . Bacteroides strains were propagated anaerobically on BHC medium [37 g of brain heart infusion base[Difco Laboratories, Detroit, Mich.] per liter, with 0.1 mgof vitamin K per liter, 0.5 mg of hemin per liter, and 50 mgof L-cysteine per liter [all from Sigma, St . Louis, Mo.]].

 
DNA isolation analysis. Plasmid DNA was extracted by the alkali lysis method [32] orusing QIAGEN columns [QIAGEN, Valencia, Calif.] . Plasmids andcosmids were restriction mapped by Southern blot walking asdescribed previously [8] . Purification of DNA fragments andextraction from gel slices were performed with a QIAEX II gelextraction kit [QIAGEN] . PCR products were cloned onto the pGEM-TEasy vector [Promega, Madison, Wis.] according to the instructionsof the manufacturer.

Colony blot hybridizations. Colony blots of B . fragilis strains were prepared by the techniquedescribed previously [8] . Briefly, B . fragilis organisms grownovernight on BHC agar were transferred to Whatman 541 filters.The filters were microwave processed in alkali solution [0.5M NaOH, 1.5 M NaCl] followed by neutralization in 2 M ammoniumacetate . The probes were labeled with [-³²P]dCTP by random priming[Multiprime DNA labeling system; Amersham Pharmacia Biotech],hybridized at 37°C under high-stringency conditions in 50% formamide-5x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate]-0.1% sodium dodecyl sulfate-1 mM EDTA-1x Denhardt's solution and washed with 5x SSC-0.1% sodium dodecyl sulfateat 65°C for 1 h . Finally, the filters were rinsed with 2xSSC at room temperature.

Construction and screening of the cosmid library. Chromosomal DNA from ETBF strain 86-5443-2-2 was partially digested with Sau3A1 under conditions where the majority of fragmentswere 30 to 40 kb in size, and it was ligated into cosmid vectorpHC79 digested with BamHI . Ligated DNA was packaged into lambdaphage by using the Giga Pack II packing extract [Stratagene,La Jolla, Calif.] and transduced into Escherichia coli HB101.The cosmid library was screened to find the right end of CTn86by using probe 11 [see Results] . Screening of the cosmid librarywas performed by colony blot hybridization as described above.

PCR conditions. The sequences of the primers and the parameters used for eachPCR are shown in Table 2 . PCRs were performed with Taq polymerase[1.5 U] in a 50-µl volume containing plasmid [5 to 10ng] or chromosomal DNA [20 ng] as template, primers [25 pmol],deoxynucleoside triphosphates [200 µM], and MgCl₂ [1.5mM].

 
Inverse PCR. Chromosomal DNA from ETBF strain 86-5443-2-2 was restrictiondigested with an appropriate enzyme [PstI or HindIII] [see Fig.3, below] . The restriction fragments [final concentration, 2.5ng/µl] were ligated to form circular fragments using 4U of T4 DNA ligase/µl . A total of 40 ng of ligated DNAwas used as a template for amplification using primers that specifically amplified the left end of CTn86 [see Fig . 2A and B,below] . The sequence and relative location of the primers as well as the parameters used for each inverse PCR are shown in Table 2 . PCRs were performed with elongase enzyme mix [Invitrogen,Carlsbad, Calif.] [1 µl] or Taq polymerase [1.5 U] whenprimers El1.2 and El3 or 86CTn1R and 86CTn2 were used, respectively.Reactions were in 50-µl volumes containing primers [25 pmol], deoxynucleoside triphosphates [200 µM], and MgCl₂ [1.5 mM].

 
Susceptibility to antibiotics. Using serial twofold dilutions, MICs of tetracycline and virginiamycinM [streptogramin A], as well as of norfloxacin and moxifloxacin[fluoroquinolones], were determined for strains 86-5443-2-2[pattern I], TM4000 [pattern II], and NCTC 9343 [pattern III]grown anaerobically in BHC medium . MICs were read after 24 hof incubation at 37°C.

Identification of CTn9343 and CTn86 intermediate circular forms. To detect the joined ends of the excised CTn9343 and CTn86,primers were designed from the ends of the CTns [Tn25A and Tn22for CTn9343, and 86CTn2 and Tn22 for CTn86] [Table 2] . These primers are directed out from the ends of the CTns and cannotyield a PCR product unless the element is in circular form.ETBF 86-5443-2-2 and NTBF NCTC 9343 and I-1345 were grown overnightin BHC broth with and without virginiamycin M [2 µg/ml],moxifloxacin [0.08 µg/ml], or norfloxacin [4 µg/ml].ETBF 86-5443-2-2 and NTBF I-1345 were also grown in BHC brothcontaining tetracycline [1 µg/ml] . Cell concentrationwas determined by densitometry after overnight growth . Similarconcentrations of cells [optical density at 600 nm, 0.8] were spun down, and 10 µl of the pellet was used as a templatefor PCR [50-µl final volume] . PCR conditions were selectedto permit detection of the PCR products in the linear rangeof the reaction [Table 2].

RT-PCR. Expression of CTn9343 genes was determined by reverse transcription-PCR[RT-PCR] [as previously described [10]] after growing strainNCTC 9343 in BHC broth and BHC broth containing virginiamycinM [2 µg/ml], moxifloxacin [0.08 µg/ml], or norfloxacin [4 µg/ml] and strain I-1345 in either BHC broth or BHCbroth containing tetracycline [1 µg/ml] . Briefly, totalRNA of overnight cultures was obtained using TRIzol reagent[Gibco BRL] according to the manufacturer's protocol . The sameamount of total RNA [4 µg] from NCTC 9343 and I-1345 grownin BHC and BHC with antibiotics was used to synthesize cDNAby using the SuperScript preamplification system for first-strandcDNA synthesis kit [Gibco BRL] following the instructions ofthe manufacturer . The cDNA samples were PCR amplified usingprimers Tn25D and Tn25, Tn25E and Tn25C, Tn1 and Tn1A, TranF and TranR, Tn18 and Tn19, and Tn21A and TnTn21 to identify expression of tnpA1, int2, rteA, traN, traG, and prmN1, respectively . Sequencesand PCR conditions are described in Table 2.

Nucleotide sequence analysis. Recombinant plasmids and PCR products were sequenced by theDNA Analysis Facility of Johns Hopkins University with an AppliedBiosystems model 373A version 2.0.S dye terminator automatedsequencer . DNA and amino acid sequences were analyzed usingthe NCBI BLAST server [1] and the Sequence Analysis softwareDNAMAN version 5.2.9 [Lynnon BioSoft, Quebec, Canada].

Nucleotide sequence accession number. The accession number of the 25-kb region containing the leftend of CTn86 and the flanking region is AY372755, and that forthe 6-kb region containing the right end of CTn86 and the flankingregion is AY375536.

 
Identification of CTn9343. To identify the complete sequence of the genetic element flankingthe BfPAI, we aligned the partial sequence [12 kb in total]of the genetic element flanking the BfPAI from ETBF 86-5443-2-2with the sequence of B . fragilis strains NCTC 9343 [patternIII; lacks the BfPAI but contains its flanking region] and 638R[pattern II; lacks both the BfPAI and its flanking region] producedby the Wellcome Trust Sanger Institute [http://www.sanger.ac.uk/Projects/B_fragilis/]. This identified that the 12-kb region sequenced in ETBF 86-5443-2-2 was 96% identical to the corresponding sequence in NCTC 9343. The ends of the flanking element then were determined by alignmentof the appropriate sequences of strains NCTC 9343 and TM4000.These alignment results initially identified that the geneticelement flanking the BfPAI was 80 kb in length and contained66 putative open reading frames [ORFs] [Table 3] . Sequence analysisusing the NCBI BLAST server [1] suggested that this geneticelement is a CTn [herein designated CTn9343].

 
CTn9343 is organized in a modular fashion, with clusters ofgenes with related functions [Fig . 1A] . Similar to CTnDOT, CTn9343 contains the regulatory genes rteA and rteB [ORFs 13 and 14, respectively]; however, these genes are not adjacent to tetQ as in CTnDOT but rather are adjacent to ORFs that predict proteins with significant homology to proteins involved in resistanceto virginiamycin M [satG] and fluoroquinolones [bexA] [Fig. 1A; Table 3] . Putative genes involved in integration-excision,like integrases [int1 and int2], transposases [tnpA1, tnpA2,and tnpB], and primases [prmN1] [Table 3] are located at the left and right ends of CTn9343 . The predicted protein encoded by int2 shares significant homology to the integrase of CTnDOT [Table 3] . Adjacent to the right-end putative integration-excisionregion, there is a cluster of genes potentially involved inthe transfer of the transposon . However, only four ORFs [ORFs36, 38, 41, and 42] of this cluster of genes predict proteins with significant homology with transfer proteins encoded by CTnDOT [Table 3] . Besides rteA, rteB, int2, and these four putativetransfer genes, there is no additional sequence homology betweenCTn9343 and CTnDOT-related transposons . In contrast to mostself-transmissible elements, the putative oriT-mob region [bfmAand bfmB] is not adjacent to the transfer region . In CTn9343,two clusters of genes [ORFs 20 to 24 and 25 to 29] and a regioncontaining ORFs 30 to 34 separate the oriT-mob from the transferregion [Fig . 1A] . The cluster containing ORFs 25 to 29, allpredicted to be transcribed in the same direction, may be involvedin the regulation of xylose utilization . The second clustercontaining ORFs 20 to 24 is also predicted to be transcribedin the same direction, although no overall potential functionfor this region is evident . The last ORF of this cluster [ORF20 [bfmC]] is a putative mobilization gene that encodes a proteinpredicted to share significant homology with a component of a type IV secretion system [Table 3] . ORFs 30, 31, and 32 arepredicted to encode proteins potentially involved in bile hydrolysis,protection from restriction endonucleases, and chromosome partitioning,respectively . Both putative ends of CTn9343 [ORFs 6, 62, 65,and 66] contain genes encoding proteins with significant homologyto proteins conferring protection from restriction endonucleasecleavage.

 
The putative start codons of ORFs 1 and 66 are located in bp 1,804,281 and 1,727,162 of the NCTC 9343 chromosome, respectively. Alignment of the NCTC 9343 and 638R sequences indicated thatthe putative left end of CTn9343 is approximately 164 bp upstreamof ORF 1, and the putative right end is 1,329 bp upstream ofORF 66 . The closet ORF to the putative left end of CTn9343 isan ORF [designated ORF-A] of 915 bp located 471 bp upstreamof ORF 1 [Fig . 1A] . The predicted protein encoded by ORF-A sharessignificant identity [73%] with a protein of unknown functionencoded by an ORF located upstream of a capsular biosynthesislocus [15] . Like this locus, 1 kb upstream of ORF-A there isa cluster of genes that may be involved in the biosynthesisof capsular polysaccharide . The closest ORF upstream of theputative right end of CTn9343 is located 1,353 bp upstream ofORF 66 [Fig . 1A] . This ORF, designated ORF-B, encodes a proteinof 310 amino acids with no significant identity to any proteinin the GenBank database.

Contribution of satG and bexA to antibiotic resistance in strain NCTC 9343. To determine if satG and bexA confer resistance to virginiamycinM and fluoroquinolones, respectively, the MICs of virginiamycinM as well as of norfloxacin and moxifloxacin [fluoroquinolones]were determined . Colony blot hybridizations using a fragmentcontaining satG and bexA as a probe [Fig. 1B, probe 5] determinedthat strains 86-5443-2-2 [pattern I; contains a genetic elementrelated to CTn9343 flanking the BfPAI] and TM4000 [pattern II;lacks CTn9343 or a related genetic element] lack satG and bexA.Notably, the MICs for strains NCTC 9343, 86-5443-2-2, and TM4000of virginiamycin and moxifloxacin were identical [10 and 0.15µg/ml, respectively], whereas 86-5443-2-2 had a higherMIC of norfloxacin [16 µg/ml] than strains NCTC 9343 andTM4000 [8 µg/ml] . These results indicate that the presenceof satG and bexA in CTn9343 does not increase resistance tovirginiamycin M and/or fluoroquinolones in strain NCTC 9343.

The BfPAI is contained in CTn86. Our laboratory's previous studies demonstrated that in ETBFstrains the BfPAI is integrated between bfmB and bfmC [9] . We sequenced a 12-kb region flanking the BfPAI in ETBF strain 86-5443-2-2.An alignment of this sequence flanking the BfPAI with the appropriatesequence of CTn9343 showed that these two sequences were 96%identical [Fig. 1B] . To determine whether the entire CTn9343 sequence flanks the BfPAI in strain 86-5443-2-2, 15 sets ofprimers spanning the entire CTn9343 sequence were designed [Table 2] . Using these primers for PCR analysis and colony blot hybridizations[using the PCR products as probes], CTn9343 was characterizedin ETBF 86-5443-2-2 [Fig . 1B] . ETBF 86-5443-2-2 was PCR andprobe positive to the central region of CTn9343 spanning probes6 to 11; however, it was negative by PCR and hybridization tothe regions spanning probes 1 to 5 and 13 to 15, suggestingthat the ends of CTn9343 in ETBF 86-5443-2-2 were deleted . ETBF86-5443-2-2 was probe positive but PCR negative to the region spanning probe 12, suggesting that the deletion of the end of CTn9343 occurred in this region . Because the CTn in ETBF 86-5443-2-2 differs from that of NCTC 9343, the genetic element flankingthe BfPAI in ETBF 86-5443-2-2 was termed CTn86 . None of the15 probes derived from CTn9343 to characterize CTn86 [Fig . 1B] hybridized with B . thetaiotaomicron 4107, a strain that contains CTnDOT integrated into the chromosome [data not shown].

The left end of CTn86 is deleted. Because CTn86 was probe negative to the region spanning probes1 to 5, the left end of CTn86 was hypothesized to be close tothe sequenced region oriT-mob [Fig . 1B] . To identify the leftend of CTn86, the sequence adjacent to the oriT-mob region wascloned by inverse PCR . Using primers El1.2 and El3 derived fromthe oriT-mob region, the next PstI fragment [4.3 kb] was clonedinto pGEM-T to originate phol.1 [Fig . 2A] . The 4.3-kb PstI fragment hybridized with all pattern I and III strains but not with any of the pattern II strains listed in Table 1, indicating that this fragment did not contain the end of CTn86 [data not shown]. Sequence analysis of the 4.3-kb PstI fragment showed that in CTn86, ORF 17 is adjacent to ORF 9 [tnpA1], with a deletionof the region that encodes int1, ORF 11, ORF 12, rteB, rteA, satG, and bexA [Fig . 2A; see also Fig. 8, below].

 
To identify the next adjacent region of the left end of CTn86,a second inverse PCR was performed to clone the next HindIIIfragment [Fig . 2B] . Unexpectedly, two PCR products, 3.0 and 1.6 kb, were obtained . Both PCR products were cloned in pGEM-Tto produce pC19-1 and pC63-1 for the 3.0- and 1.6-kb PCR products, respectively . Both PCR products hybridized by colony blottingwith all pattern II and III B . fragilis strains tested [Table 1], suggesting that both PCR products contained the left end of CTn86 [data not shown] . Sequence analysis of pC19-1 and pC63-1 revealed that ETBF 86-5443-2-2 contains two copies of CTn86 integrated in different regions of the chromosome . The sequence analyses also indicated that ORF 8 [tnpB] is the last gene in the left end of both CTn86 copies [Fig . 2B; see also Fig. 8].Comparison of the sequence of the left end of CTn86 with theappropriate sequence of CTn9343 revealed that these sequencesshared only 76% identity in the region encoding ORF 9 [tnpA1] and ORF 8 [tnpB] [Fig . 2C].

Identification of the CTn86 right end. The colony blotting and PCR results indicated that ETBF 86-5443-2-2was positive to probe 12 but negative to PCR when primers forprobe 12 were used [Fig. 1B] . These results suggested that theright end of CTn86 is contained in the region spanning probe12 . To clone the right end of CTn86, probe 11 was used to screena cosmid library of ETBF 86-5443-2-2 . A positive cosmid [19D8]was restriction mapped, and three adjacent restriction fragments[probes A, B, and C] [Fig. 3] were used to probe a collectionof B . fragilis strains [Table 1] . Probe C, but not probes A and B, hybridized with all pattern II B . fragilis strains [data not shown], indicating that the region spanning probe C contains the right end of CTn86 . Sequence analysis of the region spanning probe C revealed that ORF 61 [tnpA2] is the last ORF of the right end of CTn86 . Since ORF 61 is downstream of the predicted right end of CTn86 [the region spanning probe 12], the DNA regions spanning probes A and B were also sequenced . Comparison of the sequences of probes A to C with the appropriate sequence ofCTn9343 showed overall sequence identity of 95%; however, CTn86has deletions and additions that altered its coding sequence[Fig . 3] . CTn86 has a deletion of 446 bp containing the majorpart of ORF 57 . This deleted region is part of probe 12 andexplains the negative PCR, positive DNA hybridization resultsdiscussed above . Furthermore, ORF 58 of CTn9343 is not presentin the CTn86 sequence . In this region, CTn86 encodes two ORFs[designed ORF 58A and 58B] of 444 and 612 bp, respectively.The predicted proteins encoded by ORF 58A and 58B do not sharesignificant identity or similarity with any protein in the GenBankdatabase . The DNA region downstream of ORF 60 [500 bp] alsodiffers in CTn9343 and CTn86, and CTn86 contains an extra 730-bpsequence immediately downstream of ORF 61 [Fig . 3].

Identification of CTn86 integration sites. Sequence analysis of cosmid C19D8 [which contains the CTn86right end and 1,700 bp flanking this region] [Fig . 3] showedthat 173 bp downstream of the putative tnpA2 start codon thereis an ORF of 1,365 bp that encodes a predicted protein sharingsignificant homology [52% identity and 72% similarity] withputative Na⁺-driven multidrug efflux pump proteins . Due to thepredicted similar function of this ORF to the BexA protein describedfor B . thetaiotaomicron [18], this ORF was designated bexB [Fig. 4A] . Sequence analysis of pC19-1 [which contains the CTn86 leftend and 1,900 bp flanking this region] [Fig . 2B] revealed that the left end of one copy of CTn86 is flanked by an ORF thatencodes a predicted protein with significant homology [38% identityand 59% similarity] to ABC transporter-permease proteins . ThisORF was designated per [Fig . 4A] . Alignment of the left and right sequences flanking CTn86 with the appropriate sequenceof NCTC 9343 [http://www.sanger.ac.uk/Projects/B_fragilis/] revealed that in strain NCTC 9343 per is 2,295 bp in length [in pC19-1, only 1,900 bp were sequenced], and per and bexBare adjacent [99% identical to the region flanking CTn86] . Alignmentof the ends of CTn86 and flanking regions with the NCTC 9343per and bexB genes showed also that integration of CTn86 interruptsthe last 8 bp at the 3' end of per [Fig . 4A] . The per and bexBgenes transcribe in opposite directions, and the start codon of per is located at bp 2,587,491 of the NCTC 9343 chromosome.

 
To determine the integration site of the second copy of CTn86,the sequence of pC63-1 [which contains the CTn86 left end and 245 bp of its flanking region] [Fig . 2B] was analyzed . To determinethe locus where CTn86 is integrated, the 245-bp region flankingCTn86 was aligned with the NCTC 9343 sequence [http://www.sanger.ac.uk/Projects/B_fragilis/]. The alignment results indicated that the left end of CTn86 is flanked by an ORF of 1,122 bp encoding a protein with significant homology [73% identity and 83% similarity; score, 521] withSmF/DprA family proteins [accession no. AAO78176.1] [Fig . 4B].In Haemophilus influenzae and Helicobacter pylori, these proteinsare required for natural chromosomal and plasmid transformation[2, 13] . This ORF flanking CTn86 was designated dprA [Fig. 4B].The right end of the second copy of CTn86 is flanked by an ORFof 1,020 bp [designated ORF-C] that encodes a protein sharingsignificant homology [62% identity and 74% similarity] witha protein of B . thetaiotaomicron of unknown function [accessionno. AAO78167.1] [Fig . 4B] . ORF-C and dprA transcribe in thesame direction, and the start codon of ORF-C is bp 5,173,855 of the NCTC 9343 genome . Integration of CTn86 between dprA and ORF-C was confirmed by PCR using primers derived from the left and right chromosome-CTn86 junction [primers 86CTn8 and 86CTn2for the left end and Tn22 and 86CTn9 for the right end [Fig. 4B]] . Sequencing of the PCR products revealed that the region flanking CTn86 in ETBF 86-5443-2-2 shares 99% identity withthe appropriate sequence of NCTC 9343, and integration of CTn86 interrupts the first 5 bp at the N-terminal region of ORF-C[Fig. 4B].

CTn86 forms a circular intermediate. CTns initiate conjugal transfer by excising from the chromosometo form a circular intermediate . CTn86 excision and formationof a circular intermediate were evaluated by PCR using primersdesigned from the end sequences of the integrated transposonand directed outward [primers 86CTn2 and Tn22] . A 1.5-kb PCRproduct was detected intracellularly after growing ETBF 86-5443-2-2overnight in BHC . Identification of the CTn86 circular formsuggested that this genetic element can be transferred betweenB . fragilis strains . Strain 86-5443-2-2 is tetracycline resistant[MIC, 10 µg/ml]; however, CTn86 does not contain the tetQgene . To determine if tetracycline induces formation of theCTn86 circular intermediate, ETBF 86-5443-2-2 was grown overnightin BCH medium containing tetracycline [1 µg/ml] followedby semiquantitative PCR for the circular form . The PCR productquantity was similar after growth in the presence or absence of tetracycline [data not shown], indicating that excision and formation of the circular intermediate were not induced by tetracycline, like that of CTnDOT.

Proposed model for CTn86 integration. Sequence analysis of the 1.5-kb PCR product revealed that theexact CTn86 left end is 38 bp downstream from the putative stopcodon of ORF 8 [tnpB] and the right end is 74 bp upstream fromthe putative start codon of ORF 61 [tnpA2] . In the circularform, these nucleotides are joined by a TA sequence [Fig . 5A].The ends of the transposon contain short inverted repeat [IR]sequences of 4 bp separated by the TA sequence [Fig . 5A] . Sequence analysis of the CTn86 integrated form showed that the ends ofboth copies of CTn86 are flanked by direct repeat sequencesof 7 bp [Fig. 5C], suggesting that these direct repeat sequences are the target integration sites of CTn86 and that integrationof the transposon duplicates the target sites . The 7-bp targetsites were identified at the carboxy terminus of per and theN terminus of ORF-C . However, due to duplication of the targetsite, the start codon of ORF-C was not affected [Fig . 5B and C]. Comparison of the sequences of the ends of CTn86 with the7-bp target sites and comparison of the sequences of the 7-bptarget sites for both copies of CTn86 did not reveal any sequencesimilarity . These results suggest that integration of CTn86is not site selective.

 
The real ends of CTn9343 are tnpB and tnpA2. Initial attempts to identify the joined ends of the circularform of CTn9343 by PCR using primers derived from ORF 1 [putativeleft end] and ORF 66 [putative right end] were unsuccessful.Colony blot hybridization of our collection of B . fragilis strainsusing the 15 probes spanning CTn9343 to characterize CTn86 [Fig. 1B] showed that only 8 of 80 NTBF pattern III strains [expectedto contain CTn9343] were positives to probes 1 to 3 and 13 to15 [Franco and Sears, unpublished] and, unexpectedly, 2 of 89 B . fragilis pattern II strains [expected to lack CTn9343 and CTn86 transposons] were positive to probes 1 to 3 and 13 to15 [strains LM46 and K518 [Table 1]] . BLASTP [1] analysis ofthe ORFs contained within the regions spanning probes 1 to 3and 13 to 15 showed that ORF 5 encodes a protein with significanthomology to integrases [designated int1], and ORFs 6, 7, 62,65, and 66 encode proteins with homology to components of a type I restriction modification system [type I R-M system] [Table 3] . Proteins encoded by ORFs 6 and 62 had significant homologywith subunit S [hsdS] of the type I R-M system, and proteinsencoded by ORFs 65 and 66 had significant homology with subunitsM [hsdM] and R [hsdR], respectively [Table 3; Fig . 6] . Becauseof these results and the results defining CTn86, the ends ofCTn9343 were hypothesized to be adjacent to ORF 8 [tnpB] andORF 61 [tnpA2], similar to CTn86, and the DNA regions flankingthese ORFs [ORFs 1 to 7 at the left end and 62 to 66 at theright end, encompassed by probes 1 to 3 and 13 to 15] were hypothesizedto represent another foreign genetic element.

 
To address this latter hypothesis, primers Tn24 and Tn23 derivedfrom ORF 7 and ORF 63 sequences [Fig . 6], respectively, were used for PCR with the two B . fragilis pattern II strains positive by hybridization to probes 1 to 3 and 13 to 15 [strains LM46 and K518 [Table 1]] . The PCR yielded a product [ca . 1.5 kb] in both B . fragilis pattern II strains but not in strains NCTC 9343 or TM4000 [pattern II strains negative to probes 1 to 3 and 13 to 15], suggesting that the regions spanning probes 3and 13 are adjacent in these two pattern II B . fragilis strains[Fig. 6] . Sequence analysis of the PCR product identified an ORF [designated hsdS] . The first 1,343 bp of this ORF had significantidentity [93%] to ORF 62, and the last 129 bp shared significantidentity [99%] with ORF 7 . These results indicated that CTn9343is contained in another foreign genetic element encoding a typeI R-M system, and its integration in this genetic element interruptsthe hsdS gene . Our results also indicate that, like CTn86, theleft and right ends of CTn9343 are close to the stop codon oftnpB and start codon of tnpA2, respectively [Fig. 6; see alsoFig . 8].

Identification of the CTn9343 intermediate circular form. Once the likely real ends of CTn9343 were determined, primersderived from the left [Tn25A] and right [Tn22] ends were designedto detect the intermediate circular form . Similar to ETBF 86-5443-2-2,a 0.8-kb PCR product was detected intracellularly after growingNCTC 9343 overnight in BHC broth, suggesting that CTn9343 maybe transferable between B . fragilis strains.

To determine if virginiamycin M and/or fluoroquinolones induce formation of the CTn9343 circular intermediate, strain NCTC9343 was grown overnight in the presence of virginiamycin Mas well as norfloxacin and moxifloxacin [fluoroquinolones].No induction of NCTC 9343 was detected by PCR in the presenceof these antibiotics [data not shown] . Furthermore, RT-PCR analysisshowed similar levels of expression of rteA, putative integration-excisiongenes int2, tnpA1, and prmN1, and putative transfer genes traN and traG after growth of strain NCTC 9343 in the presence or absence of these antibiotics [data not shown] . These results suggested that virginiamycin M and/or fluoroquinolones do notinduce formation of the CTn9343 circular intermediate . StrainNCTC 9343 is tetracycline sensitive, and so induction of theCTn9343 circular form by tetracycline was determined using thetetracycline-resistant strain I-1345 [10] . Similar to NCTC 9343,strain I-1345 is pattern III and hybridizes with the 15 probesderived from different regions spanning CTn9343 [Fig . 1B] . These results suggest that strain I-1345 contains a genetic elementsimilar to CTn9343 . The CTn9343 circular form was detected instrain I-1345, and the circular form was not induced by tetracycline[data not shown] . Similarly, RT-PCR analysis showed that tetracyclinedid not induce expression of rteA, int2, tnpA1, prmN1, traN,or traG in strain I-1345 . These results suggest that tetracyclinedoes not induce CTn9343 excision and formation of circular intermediates.

Proposed model for CTn9343 integration. Sequence analysis of the 0.8-kb PCR product to identify thecircular form revealed that the exact ends of CTn9343 are 89bp downstream from the putative stop codon of ORF 8 [tnpB] and74 bp upstream from the putative start codon of ORF 61 [tnpA2].These bases are joined by an AT sequence and, like CTn86, theends of CTn9343 have a short IR sequence of 4 bp separated bythe AT sequence [Fig . 7A] . Similar to CTn86, the target siteof CTn9343 is a 7-bp sequence that is duplicated after the integrationof the transposon [Fig. 7B and C] . The 7-bp target site of CTn9343does not share any similarity with the target sites of CTn86or with the ends of CTn9343, suggesting that integration ofCTn9343 is not site specific.

 
In this study, the genetic element flanking the BfPAI in ETBF 86-5443-2-2 and a related genetic element in NCTC 9343 were identified and characterized . The results suggest that thesegenetic elements are members of a new family of CTns not described previously . These putative CTns, designated CTn86 and CTn9343,for ETBF 86-5443-2-2 and NCTC 9343, respectively, differ frompreviously described Bacteroides species CTns in a number ofways . These new transposons do not carry tetQ, and the excisionand formation of circular intermediates are not regulated by tetracycline; they are predicted to have a different mechanismof transposition; and their sequences have very limited sequence homology with CTnDOT or other described CTns.

Initial alignment results with NCTC 9343 [pattern III] and 638R [pattern II] sequences indicated that CTn9343 was 80 kb in length;however, further colony blot hybridizations, PCR, and sequenceanalysis determined that the real ends of CTn9343 define a geneticelement of 64,229 bp . The results indicated that the 16-kb regionflanking CTn9343 is another foreign genetic element that encodesa type I R-M system [43] and a lambda family integrase [int1][24] . The highest homologies of the putative proteins comprisingthis type I R-M system [HsdS, HsdR, and HsdM], as well as Int1,are with those of Streptococcus pneumoniae [Table 3], suggesting that B . fragilis strains may have acquired this genetic element by horizontal transfer from a gram-positive organism.

CTn9343 may also have arisen from gram-positive bacteria . CTn9343 harbors antibiotic resistance genes [fluoroquinolones and virginiamycin M] that are similar to those from gram-positive organisms . The predicted protein encoded by the virginiamycin M resistancegene [satG] is 65% identical and 79% similar to an analog proteinof Clostridium acetobutylicum [Table 3] . Virginiamycin M is usually used in animal feed; this factor may have selected for the evolution and transfer of CTn9343 . Recently, a new Bacteroides CTn, CTnGERM1, has been reported that also contains genes found in gram-positive bacteria [39] . These results support the ideathat CTns can be acquired by Bacteroides species from othergenera . B . fragilis strains may have acquired CTn9343 and itsflanking genetic element together during the transposition of CTn9343 or, alternatively, B . fragilis strains may have first acquired the genetic element encoding a type I R-M system and then it was interrupted by integration of CTn9343.

The MIC results suggested that the B . fragilis strains tested are intrinsically resistant to virginiamycin M, and the presence of satG did not increase the resistance to this antibiotic in strain NCTC 9343 . Similarly, the presence of bexA did not increase resistance to the tested fluoroquinolones . The predicted protein encoded by bexA shares significant homology with a multidrug efflux transporter involved in fluoroquinolone resistance in B . thetaiotaomicron [18] . A portion of the norfloxacin resistanceof B . fragilis and B . thetaiotaomicron is attributed to activeefflux of the antibiotic by BexA [17, 18] . However, BexA maynot be functional in CTn9343, since the protein lacks 200 N-terminalamino acids in comparison to the analog protein encoded by B.thetaiotaomicron.

The structure of CTn9343 appears to be modular, containing genes derived from different bacteria, bacteriophages, viruses, plasmids, and unknown sources [Table 3] . The G+C content of the entire CTn9343 sequence [46.5%] is different from that reported for the B . fragilis chromosome [43% [http://www.sanger.ac.uk/Projects/B_fragilis/]]. However, there are several CTn9343 regions whose G+C contents differ significantly from the rest of the transposon . For example, the putative transfer region [see below] has a G+C content of53%, and the region containing the rteB, rteA, satG, and bexA genes has a G+C content of 45% . These results suggest that CTn9343 originated from different genetic sources . In contrast, theG+C content [43%] of the cluster of genes [ORFs 25 to 29] encodingthe enzyme for utilization of xylose together with an ECF-typesigma factor and two-component systems [Table 3] is characteristic of the genome of Bacteroides species [46], suggesting that CTn9343also contains genes of Bacteroides species origin.

There is limited homology between CTn9343, CTnDOT, and related CTns . None of the DNA probes used to characterize CTn86 [Fig. 1B] hybridized with CTnDOT under the conditions tested [see Materials and Methods] . Only the region of CTn9343 containing int2-rteB-rteA and four putative transfer genes [ORFs 36, 38,41, and 42] have homology with CTnDOT . This may indicate that these regions were acquired by CTn9343 and CTnDOT from a common ancestor . The putative integrase [Int2] encoded by CTn9343 has significant homology with the integrase of CTnDOT [Table 3]; however, the integrase of CT9343 lacks 128 amino acids fromthe N terminus and 188 amino acids from the carboxy terminus[this includes the region termed box 2, which is conserved inthe lambda integrase family and is considered important to integrasefunction] of CTnDot . In the lambda integrase family, the carboxyterminus, missing in the CTn9343 Int2, contains the catalyticsite that carries out the DNA breaking and joining reactionsthat mediate recombination [24].

In the case of CTnDOT, the integrase gene and regulatory rteA, rteB, and rteC genes together with the exc gene are essentialfor excision and integration [5, 6, 42] . The CTnDOT rteA and rteB genes together with the tetracycline resistance gene tetQ form an operon . RteA and RteB are members of a two-component regulatory system; however, recently it has been reported that neither RteA nor RteB affects expression of the tetQ-rteA-rteB operon [40] . This operon may be regulated by TetQ by a translationalattenuation mechanism in response to tetracycline [40] . In contrastto CTnDOT, CTn9343 lacks tetQ, and rteA and rteB are in a putativeoperon that also includes satG and bexA . Exposure of NCTC 9343cells to virginiamycin M or fluoroquinolones [antibiotic resistanceencoded by satG and bexA, respectively] as well as tetracyclinedid not increase formation of the CTn9343 circular intermediateor expression of rteA or putative genes involved in the excision-integrationor transfer of CTn9343 . These results suggested that the rteB/rteA/satG/bexA operon does not regulate excision and transfer of CTn9343 . The rteB/rteA/satG/bexA operon may not be functional in CTn9343,because rteA and bexA encode truncated proteins . RteA lacks349 amino acid residues from the carboxy-terminal end in comparisonto RteA of CTnDOT, and BexA lacks 200 amino acids from the Nterminus.

The homology of ORFs 36, 38, 41, and 42 to CTnDOT transfer proteins TraN, TraM, TraI, and TraG and the location of these ORFs ina large cluster of genes [ORF 35 to 50], all of which [exceptORF 37] are predicted to be transcribed in the same direction,suggest that this region is the transfer region of CTn9343.Beside these four transfer proteins, there is no additionalsequence similarity between the CTn9343 putative transfer regionand other conjugative elements . It has been reported that thetransfer region of CTnDOT is totally different from that ofgram-positive and gram-negative organisms [3] . Only TraG sharessequence similarity with proteins encoded by other transmissibleelements [3] . The presence in CTn9343 of genes with similarityto traN, traM, traI, and traG genes may indicate that the proteinsencoded by these genes are absolutely necessary for conjugaltransfer in Bacteroides species.

Colony blot hybridization, PCR, and sequence analysis indicated that CTn86 has a structure similar to CTn9343, except that CTn86 lacks an 7-kb region identified in CTn9343 containing int2,ORF 11, ORF 12, rteB, rteA, satG, and bexA and it contains the BfPAI [Fig . 8] . The G+C content of the BfPAI [35%] also differsgreatly from that of other regions of CTn86, suggesting acquisitionfrom a different organism.

Even though the region encoding int2 and the regulatory genes, rteA and rteB, has been deleted in CTn86, this transposon formscircular intermediates . These results further suggest that the truncated Int2 and RteA proteins are not functional in CTn9343, and other genes in the transposon regulate the transposition. Analysis of the ends and flanking regions of CTn9343 and CTn86in the circular and integrate forms [Fig . 5 and 7], respectively,suggests that these transposons integrate in a similar way toIS21 . IS21 is bordered by IRs of different lengths and containstwo contiguous genes, istA and istB, forming an operon [16].The hallmark of the IS21 transposition mechanism is the spontaneousformation of IS21 tandem repeats, designated [IS21]₂ [27] . In the [IS21]₂ configuration, the two insertion sequences are typicallyseparated by 2 or 3 bp, termed a junction sequence [26] . Thetandemly repeated copies of IS21 promote insertion of entireplasmids carrying [IS21]₂ in a transposition event involvingthe abutted terminal IR sequences [26] . During this cut-and-pasteprocess, the junction sequence of [IS21]₂ is lost, and the outerends of IS21 are dispensable . The cointegrates formed have asingle IS21 copy at each junction, and the IS21 elements arebordered by direct repeats of 4 bp, i.e., the target duplication[26].

The left ends of CTn9343 and CTn86 have two consecutive genes, tnpA1 and tnpB, that predict encoded proteins with significant homology to IS21 IstA and IstB, respectively . IstA and IstB are required for integration [34] . The right end has a genethat encodes a truncated TnpA1 [tnpA2] . In CTn9343 and CTn86,joining of the transposon ends to form the circular intermediatesyields a structure where one copy contains tpnA1 and tnpB andthe second contains tnpA2 . The ends of both copies contain IRsequences separated by a 2-bp junction sequence [Fig . 5A and7A] . Similar to the structure of [IS21]₂, where the ends contain short IR sequences separated by 2 nucleotides, transpositionof the circular forms into the B . fragilis chromosome duplicates the 7-bp target site with loss of the 2-bp junction sequence[Fig. 5B and C and 7B and C] . The tnpA2 gene encodes a truncatedprotein of 124 amino acid residues in CTn9343 and CTn86 . Itis unknown if TnpA2 is functional in CTn9343 and CTn86 or onlyserves to join with the right end to form an active circularform.

Together, this predicted mechanism of excision and integration described for CTn9343 and CTn86 differs from that of CTnDOTor other reported CTns [5, 7, 28] . The absence of homology betweenthe 7-bp target sites with the ends of CTn9343 and CTn86, aswell as the absence of a consensus sequence in the 7-bp targetsites of CTn9343 and CTn86, suggest that, in contrast to CTnDOT,insertion of CTn9343 and CTn86 is not site specific.

In conclusion, based on sequence analysis and identificationof the circular intermediates, the genetic element flankingthe BfPAI in ETBF strain 86-5443-2-2 and a related genetic elementin strain NCTC 9343 are predicted to be CTns [CTn86 and CTn9343,respectively] . These putative CTns may be members of a new familyof CTns . These putative CTns are distinct in that CTn86 lacksa 7-kb region containing a truncated integrase [int2] and rteAgenes and possesses the BfPAI . If CTn86 were demonstrated tobe transmissible, this would suggest that the bft gene can be transferred from ETBF to NTBF strains by a mechanism similarto that for the spread of antibiotic resistance genes . Furtherstudies are necessary to determine if the transfer genes ofCTn86 and CTn9343 are functional.

 
I thank Cynthia L . Sears for review of the manuscript and helpful discussions and Janeth P . Castillo for excellent technical assistance.

This work was supported by grant number RO1A148708 [A.A.F.]from the National Institutes of Health.

 
* Mailing address: Division of Infectious Diseases, Johns Hopkins University School of Medicine, Ross Bldg., Rm . 1167, 720 Rutland Ave., Baltimore, MD 21205 . Phone: [410] 955-9686 . Fax: [410] 614-9775 . E-mail: afranco@jhem.jhmi.edu.

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